Tumor necrosis factor alpha (TNF-alpha) is a cytokine that exists as a trimeric molecule and has two bioactive forms—membrane-bound TNF-alpha and soluble TNF-alpha. The membrane bound form of TNF-alpha is composed of extracellular, transmembrane and cytoplasmic domains. Cleavage of the membrane bound form (about 26 kDa) results in the soluble form (˜17 kDa), which exists as a homodimer. While both forms are biologically active, the soluble form of TNF-alpha is more potent.
TNF-alpha exerts effects on cell proliferation, cell differentiation, inflammation, and cell death, as well as on immunoregulation, by binding to specific cell surface receptors. The groove created by neighboring subunits of the trimeric molecule is important for interaction with its receptors. The two known TNF-alpha receptors include the p55 (CD120a) and p75 (CD120b) receptors. The p55 receptor is expressed on most cells; whereas the p75 receptor shows much more restricted expression, mainly on activated white blood cells. It is believed that TNF-alpha plays an important role in various conditions and disorders, including, for example, inflammatory disorders, such as rheumatoid arthritis, psoriatic arthritis, and Crohn's disease.
Chronic inflammatory diseases can be life-changing, debilitating conditions, and rheumatoid arthritis (RA) is one of the most common. RA is characterised by symmetric inflammation of the peripheral joints, resulting in progressive destruction of the joint. Approximately 1-2% of the world's population is affected by RA, and one in three patients is likely to become severely disabled within 20 years of diagnosis. The onset of RA most often occurs between the ages of 40 and 50, with three times more women affected than men. There is no cure for RA; treatment is aimed at slowing the disease and minimizing joint damage, while maintaining quality of life.
Several strategies have been developed to antagonize the action of TNF-alpha with its receptors in various conditions where the cytokine has been implicated as a causative agent. For example, blocking the action of excessive TNF-alpha is a therapeutic strategy in several inflammatory diseases, like rheumatoid arthritis. With RA, in particular, there has been a recent shift in treatment paradigm, with new emphasis on starting treatment against TNF-alpha earlier in the course of the disease. One type of therapeutic directed against TNF-alpha involves anti-TNF-alpha antibodies.
Conventional antibodies, however, may be difficult to raise against multimeric proteins, where the receptor-binding domain is embedded in a groove, as is the case with the active forms of TNF-alpha. Furthermore, mammalian cellular systems are normally needed to express intact, functional antibodies, which contributes to high costs of manufacture. Obtaining a therapeutically useful antibody can require additional modification, such as avoiding unwanted immunological reactions upon administration to a human subject. Traditional antibodies also may not be stable at room temperature, requiring refrigeration during manufacture, storage, and transport, further adding to expense. Moreover, the large size of conventional antibodies can restrict solubility and tissue penetration.
One approach to addressing some of these issues involves the use of single domain antibodies. However, the relatively small size of such molecules can result in the products having short half lives in vivo. Several strategies have been used to overcome the problems associated with short serum half-life. For example, certain protein fusions have been attempted to increase the size and, thus, stability and half-life of protein therapeutics. (Syed et al. (1997) Blood 89:3243-3252; Yeh, et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89:1904-1908). Fusion products are prone to misfolding, however, and the fused region can create additional immunogenic sites. Nonetheless, fusions of therapeutic proteins to albumin have been attempted.
Albumin (molecular mass of 67 kDa) is the most abundant protein in plasma, present at 50 mg/ml, and having a half-life of 19 days in humans (Peters, T., Jr. (1985) Adv. Protein Chem. 37:161-245; Peters, T., Jr. (1996) All about Albumin, Academic Press, Inc., San Diego, Calif.). Albumin plays a vital role in vivo by reversibly binding and transporting a wide variety of endogenous substances as well as drugs, and several major small molecule binding sites in albumin have been described. (e.g., see, Frick, et al. (1994) Mol Microbiol. 12:143-51; and Akesson et al. (1994) Biochem. J. 300:877-886). Still a further strategy involves coupling the therapeutic to another protein that allows in vivo association to serum albumins. Examples of this approach also have been described, e.g. in EP 0486525 and U.S. Pat. No. 6,267,964, which describe the use of albumin-binding peptides or proteins derived from streptococcal protein G (SpG) for increasing the half-life of other proteins.
There are at least five anti-TNF-alpha products on the market, Infliximab (Remicade™), Adalimumab (Humira), Etanercept (Enbrel), Certolizumab pegol (Cimzia), and Golimumab (Simponi), each of which is associated with a high cost of treatment. For example, each of the anti-TNF products on the market use non-human primates as the main relevant species for assessing toxicity, contributing to high costs. More importantly, existing treatments can be marginally effective and/or poorly tolerated, while too expensive for feasible use in combinations. Approximately one-third of patients fail to respond to these therapies and adverse reactions are common. Patients who have a poor response with one TNF-alpha inhibitor are typically switched to another and, further, many patients develop an immunogenic response towards a given product and thus similarly need to change their treatment.
Accordingly, there is a need for therapeutics directed against TNF-alpha that provide specificity, solubility, and longer half-life, as well as reduced immunogenicity in the subject, in a cost-effective manner. In particular, there is a need for therapeutics to treat TNF-alpha mediated conditions, like inflammatory disorders, that can be given less frequently, at lower doses, in combination with existing drugs, and/or in a manner that facilitates self-administration. Antibody single domain-based therapeutics provided herein address these and other needs.
The foregoing discussion is presented solely to provide a better understanding of the nature of the problems confronting the art and should not be construed in any way as an admission as to prior art nor should the citation of any reference herein be construed as an admission that such reference constitutes “prior art” to the instant application.